Macrophages mediate flagellin induced inflammasome activation and host defense in zebrafish.

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Cell Microbiol. Author manuscript; available in PMC 2017 April 01. Published in final edited form as: Cell Microbiol. 2016 April ; 18(4): 591–604. doi:10.1111/cmi.12536.

Macrophages Are Required For Inflammasome-Dependent Host Defense In Vivo William J.B. Vincent1,2, Christina M. Freisinger2,3, Pui-ying Lam4, Anna Huttenlocher1,2,3,4,#, and John-Demian Sauer1,2,# 1Microbiology

Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin,

53706, USA

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2Department

of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA

3Department

of Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA

4Program

in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA

Summary

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The inflammasome is an innate immune complex whose rapid inflammatory outputs play a critical role in controlling infection, however the host cells that mediate inflammasome responses in vivo are not well defined. Using zebrafish larvae, we examined the cellular immune responses to inflammasome activation during infection. We compared the host responses to two Listeria monocytogenes strains: wild type and Lm-pyro, a strain engineered to activate the inflammasome via ectopic expression of flagellin. Infection with Lm-pyro led to activation of the inflammasome, macrophage pyroptosis, and ultimately attenuation of virulence. Depletion of caspase A, the zebrafish caspase-1 homolog, restored Lm-pyro virulence. Inflammasome activation specifically recruited macrophages to infection sites, whereas neutrophils were equally recruited to WT and Lm-pyro infections. Similar to caspase A depletion, macrophage deficiency rescued Lm-pyro virulence to wild type levels, while defective neutrophils had no specific effect. Neutrophils were however important for general clearance of L. monocytogenes, as both wild type and Lm-pyro were more virulent in larvae with defective neutrophils. This study characterizes a novel model for inflammasome studies in an intact host, establishes the importance of macrophages during inflammasome responses, and adds importance to the role of neutrophils in controlling L. monocytogenes infections.

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INTRODUCTION The innate immune response is critical for host defense against invading pathogens. Mechanisms have evolved to rapidly sense and protect the host from infection (Janeway and Medzhitov, 2002), including the activation of the multi-protein signaling complex known as

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Co-Corresponding Authors: Dr. Anna Huttenlocher, University of Wisconsin, Madison, 4205 Microbial Sciences Bldg., 1550 Linden Dr., Madison, WI 53706, USA. [email protected]; Dr. John-Demian Sauer, University of Wisconsin, Madison, 4203 Microbial Sciences Bldg., 1550 Linden Dr., Madison, WI 53706, USA. [email protected].

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the inflammasome (Martinon et al., 2009). The inflammasome is important for defense against many infections including intracellular bacterial pathogens (Sansonetti et al., 2000; Mariathasan et al., 2005; Lara-Tejero et al., 2006; Broz et al., 2010), as well as fungal and viral pathogens (Rathinam et al., 2010; van de Veerdonk and Joosten, 2015). To counter this host defense, many pathogens have evolved mechanisms to avoid or even actively inhibit inflammasome activation (Taxman et al., 2010; Lamkanfi and Dixit, 2011; Ulland et al., 2015). Despite substantial progress on the molecular mechanisms of inflammasome activation, little is known about how host protection is mediated and what the role of specific innate immune cell populations is during acute infection in vivo.

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The inflammasome surveys the cytosol for signals of intracellular pathogens or host cell damage. Canonical inflammasome activation begins with the detection of a cytosolic ligand or danger signal, which induces the self-oligomerization of a corresponding inflammasome sensor (Martinon et al., 2009), typically either nucleotide-binding domain, leucine-rich repeat containing receptors (NLRs) or AIM2-like receptors (ALRs) (Kanneganti, 2015). Following receptor oligomerization, the adaptor protein apoptosis-associated speck like protein with a caspase recruitment domain (ASC) is recruited to the activated sensor. ASC links ligand sensing to inflammation by binding to and activating the inflammatory cysteineaspartic protease caspase-1 (Martinon et al., 2002; Mariathasan et al., 2004; Faustin et al., 2007). Caspase-1 activates a number of inflammatory outputs including the activation of IL-1β, the production of eicosanoids, and a pro-inflammatory form of programmed lytic host cell death termed pyroptosis (Chen et al., 1996; Martinon et al., 2002; Mariathasan et al., 2004; von Moltke et al., 2012). The molecular mechanisms of inflammasome activation have been worked out in vitro largely using macrophages and dendritic cells, but it is not fully understood how these or other relevant cells of the immune system contribute to inflammasome-mediated host protection in vivo.

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To address this gap, we have developed a model in zebrafish to study inflammasome activation and the contributions of innate immune phagocytes to host defense. The zebrafish is a powerful model system that is highly amenable to genetic manipulation and high resolution imaging, and has a conserved innate immune system (Meijer and Spaink, 2011; van der Vaart et al., 2012). Many of the components of the inflammasome are conserved in zebrafish. For example, a family of NLRs exist in the zebrafish genome (Laing et al., 2008), although the ligands that they respond to have yet to be identified. The adaptor protein ASC is also conserved and oligomerization of zebrafish ASC recruits and activates caspase A, the zebrafish functional homolog of caspase-1, into a characteristic speck structure (Masumoto et al., 2003). Furthermore, in ex vivo leukocytes from adult zebrafish, caspase A can cleave IL-1β upon stimulation with the fish specific pathogen Francisella noatunensis (Vojtech et al., 2012). Live imaging of lytic macrophage death and hallmarks of pyroptosis have also been observed in a zebrafish larval model of Shigella flexneri infection (Mostowy et al., 2013). Infection with spring viremia of carp virus (SVCV), a fish hemorrhagic virus, leads to a sharp decline in macrophage number and induces membrane permeability. In this system caspase A and IL-1β were co-localized by immunostaining within actively infected macrophages and showed IL-1β being released from macrophages (Varela et al., 2014). Collectively, these studies provide strong evidence that inflammasome signaling is conserved in zebrafish. Cell Microbiol. Author manuscript; available in PMC 2017 April 01.

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We developed a system to probe the inflammasome in zebrafish using a localized Listeria monocytogenes infection model. L. monocytogenes is a cytosolic pathogen that upon uptake into host cells produces a pore-forming cytolysin, listeriolysin O (encoded by the gene hly), to escape the phagosome (Gaillard et al., 1987). Once in the host cytosol, L. monocytogenes rapidly replicates and makes use of an actin nucleating factor ActA (actA) to hijack the host cytoskeleton and spread from cell to cell without leaving the intracellular niche (Kocks et al., 1992). These classic steps of L. monocytogenes virulence have been observed during systemic infection of zebrafish larvae (Levraud et al., 2009). To promote virulence and protect its intracellular niche, L. monocytogenes has evolved to largely evade inflammasome activation (Sauer et al., 2010; Sauer et al., 2011; Witte et al., 2012). However, it has previously been demonstrated that L. monocytogenes and other pathogens engineered to ectopically express flagellin in the host cytosol, robustly activate the inflammasome and can be used to understand the role of the inflammasome in host defense and induction of adaptive immunity (Miao et al., 2010; Sauer et al., 2011; Warren et al., 2011). Inflammasome activation leads to rapid and robust attenuation of L. monocytogenes virulence, however, as with Salmonella typhimurium, IL-1β and IL-18 are dispensable for this phenotype (Miao et al., 2010; Sauer et al., 2011). L. monocytogenes that ectopically express flagellin (Lm-pyro) recruit LysM-positive cells to the spleen earlier than WT infection, however the inflammation also prematurely resolves (Williams et al., 2013). L. monocytogenes engineered to induce necrosis through misregulation of LLO activity are also highly attenuated, further demonstrating the importance of maintaining the intracellular replication niche (Glomski et al., 2003). Importantly, the virulence defects of necrotic L. monocytogenes strains can be rescued by neutrophil depletion (Glomski et al., 2003), however, neutrophil depletion does not rescue the virulence defect of Lm-pyro (Sauer et al., 2011), suggesting that the role of neutrophils following pyroptosis is complex. Taken together these studies suggest that IL-1β/IL-18 production and neutrophil killing are not essential for bacterial clearance due directly to inflammasome activation. The roles of other innate immune cells during this process are not fully understood.

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Here we describe a model system to examine the role of the inflammasome in the cellular innate immune response to infection. As is observed in disseminated infection models in mice, bacteria that activate the inflammasome are attenuated in a localized zebrafish infection model. Inflammasome activation leads to a substantial increase in macrophage inflammation at the site of infection and macrophages undergoing pyroptosis could be observed with high spatial and temporal resolution during infection. Leukocyte depletion supports a critical role for macrophages in inflammasome-mediated host defense. Conversely, neutrophils were important for general clearance of L. monocytogenes independent of inflammasome activation. This work establishes a tractable model for studying the innate immune response to inflammasome activation, making use of the unique features of both the zebrafish host and the intracellular pathogen L. monocytogenes, and highlights the novel finding that macrophages are essential for pathogen control in the context of inflammasome activation.

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Results L. monocytogenes hly and actA are required for localized zebrafish infection

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L. monocytogenes can establish lethal infection in zebrafish larvae following intravenous inoculation (Levraud et al., 2009). To allow for the tracking of cellular inflammatory responses to infection, we developed a hind brain ventricle (HBV) localized L. monocytogenes infection model, inoculating larvae at 48 hours post fertilization (hpf). We chose this developmental stage as both neutrophils and macrophages have developed and are phagocytically active, but are present in only small numbers at the HBV (Herbomel et al., 1999; Herbomel et al., 2001; Lieschke et al., 2001; Le Guyader et al., 2008). Following inoculation into the HBV, WT L. monocytogenes was able to cause host death at each dose analyzed, as low as 10 CFU (Figure 1A). Virulence increased in a dose dependent manner, with an apparent LD50 of 100 CFU. Mutant strains of L. monocytogenes that are unable to escape the primary phagosomal compartment (∆hly) (Gaillard et al., 1987) or maintain access to the cytosol via cell to cell spread (∆actA) (Kocks et al., 1992) were significantly attenuated at all doses examined (Figure 1B–C).

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To examine the kinetics of dissemination, we performed HBV infections using 100 CFU— the WT LD50 dose—of WT and mutant strains constitutively expressing GFP, and examined embryos for signs of disseminated infection. At 1 day post infection (dpi), no visible dissemination could be seen, likely due to low resolution imaging. Between 2 and 3 dpi, patches of WT L. monocytogenes emerged at distal points in the trunk and tail muscle along the neural tube (Figure 1D), away from the inoculation site. From 3–4 dpi these patches expanded as embryos succumbed to infection. Dissemination was not witnessed during infection with ∆hly or ∆actA L. monocytogenes (Figure 1E–F)—which looked identical to PBS inoculated larvae (Supplementary Figure 1)—nor were there signs of necrotic tissue damage as seen during WT infection. Taken together, these data suggest that localized HBV infection with WT L. monocytogenes leads to disseminated disease and ultimately death across a wide range of doses dependent upon bacterial access to the host cytosol. Lm-pyro is attenuated in zebrafish larvae

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Given that L. monocytogenes zebrafish infection required access to the cytosol, we hypothesized that other adaptations that maintain the intracellular niche are similarly important for virulence. It has previously been demonstrated that L. monocytogenes that activate host cell death are attenuated in vivo (Glomski et al., 2003; Warren et al., 2008; Sauer et al., 2010; Sauer et al., 2011; Warren et al., 2011). Specifically, Lm-pyro is genetically engineered to hyper-activate the mouse Nlrc4 inflammasome by secreting flagellin monomers exclusively when in the host cytosol (Sauer et al., 2011). Lm-pyro carries a genomically inserted construct that uses the actA promoter to drive secretion of the FlaA flagellin from Legionella pneumophila specifically when the bacterium reaches the host cytosol and initiates actA expression. In agreement with previous studies (Sauer et al., 2011; Warren et al., 2011), Lm-pyro was attenuated following inoculation at a WT LD50 dose (Figure 2A). In contrast to WT infection, the vast majority of larvae infected with Lmpyro bacteria constitutively expressing GFP did not display signs of disseminated infection (Figure 2B). Furthermore, Lm-pyro bacterial growth within larvae was decreased compared


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to WT infection (Supplementary Figure 2). Taken together, these data show that at the WT LD50 dose, secretion of flagellin into the cytosol leads to virulence defects and clearance from the zebrafish host. Despite attenuation at lower doses, WT and Lm-pyro infection were equally virulent at 1000 CFU and indeed Lm-pyro was statistically more virulent at 10000 CFU (Figure 2A). Thus, host responses downstream of sensing cytosolic flagellin can be overwhelmed with a high inoculum. Lm-pyro activates the inflammasome in zebrafish

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Lm-pyro is attenuated, both in macrophages and in vivo in mice, due to activation of the Nlrc4 inflammasome (Sauer et al., 2011). We therefore examined if Lm-pyro activates the inflammasome in zebrafish, and if so, if this activation plays a role in controlling Lm-pyro infection. To determine if flagellin, and specifically Lm-pyro, activates a zebrafish inflammasome, we generated a GFP tagged form of the zebrafish ASC, made mRNA by in vitro transcription, and used this to assay for ASC speck formation. Zebrafish larvae overexpressing GFP tagged ASC were inoculated with PBS, WT, or Lm-pyro L. monocytogenes and ASC specks at the inoculation site were quantified. Overexpression of GFP-ASC alone induced ASC specks, similar to cell culture systems (Masumoto et al., 2001; Masumoto et al., 2001). Compared to mock inoculation with PBS, infection with WT bacteria did not lead to an increase in ASC specks, consistent with a lack of inflammasome activation by WT L. monocytogenes during in vivo infection (Sauer et al., 2010; Sauer et al., 2011; Witte et al., 2012). By contrast, infection with Lm-pyro led to an increase in the number of ASC specks compared to WT bacteria (Figure 3A–B). Thus, the host response to cytosolic flagellin increased inflammasome activation as reported by ASC specks, whereas the presence of WT L. monocytogenes alone did not.


We next examined embryos actively infected with Lm-pyro for signs of pyroptosis, a programmed lytic host cell death downstream of inflammasome activation. To detect pyroptosis in vivo we generated a zebrafish line with histone-2B tagged in macrophages (Tg(mpeg1:histone2b-mCherry)). We then crossed this line to zebrafish in which macrophages were tagged with cytosolic dendra2, allowing us to image both nuclear morphology and plasma membrane lysis. Larvae were infected with either WT or Lm-pyro bacteria expressing mCherry driven by the actA promoter, allowing us to focus on actively infected macrophages. At 24 hpi, we were able to image in vivo pyroptosis in detail during Lm-pyro infection (Figure 3C and Supplementary Movie). This process was characterized by rapid loss of cytosolic fluorescence immediately followed by nuclear condensation. The total process was complete within 10–20 minutes. Multiple macrophages could be seen undergoing pyroptosis, quickly followed by increased activity of both nearby macrophages and other unlabeled cells (Supplementary Movie). Despite repeated attempts, we were unable to find any macrophages undergoing pyroptosis in WT infected embryos. Thus pyroptosis, one of the classic outcomes of inflammasome activation, was witnessed during infection with Lm-pyro. Taken together, these data suggests that, consistent with previous observations in murine systems, wild type L. monocytogenes stimulates little to no inflammasome activation in vivo, whereas Lm-pyro triggers classic hallmarks of inflammasome activation including ASC oligomerization and pyroptosis. Furthermore, this


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data suggests that zebrafish contain an intact inflammasome signaling cascade similar to the NAIP5/Nlrc4 inflammasome found in mice. Inflammasome activation attenuates Lm-pyro virulence

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The increased utilization of ASC combined with imaging pyroptosis strongly suggested that Lm-pyro was activating the inflammasome in our model system. Downstream events following inflammasome activation are ultimately executed by caspase-1. As virulence attenuation of Lm-pyro in other systems was dependent on inflammasome activation, we examined the ability of WT and Lm-pyro L. monocytogenes to cause disease following administration of morpholino oligonucleotides to transiently knock down translation of caspase A (Masumoto et al., 2003), the zebrafish homolog of caspase-1. Knockdown efficiency was verified by western blot (Supplementary Figure 3). Caspase A knockdown had no effect on the virulence of WT L. monocytogenes at the LD50 dose (Figure 4A), suggesting as previously reported (Sauer et al., 2011) that WT L. monocytogenes minimally activates the inflammasome in vivo. There was, however, a significant increase in Lm-pyro virulence following knockdown of caspase A (Figure 4B), suggesting that the attenuated virulence of Lm-pyro is due to host signaling through the inflammasome. Of note, virulence during Lm-pyro infection was not fully rescued to WT levels upon caspase A knockdown (p < 0.0001 comparing WT vs Lm-pyro infected CaspA morphants). However, this is likely due to both the incomplete knockdown of Caspase A as well as the return of Caspase A expression and hence inflammasome signaling at 4 dpf/2 dpi (Supplementary Figure 3). Together, these data suggest that, similar to mammalian systems, inflammasome activation in the zebrafish can be host protective in the context of intracellular bacterial infection. Inflammasome activation induces macrophage recruitment


Having demonstrated that Lm-pyro activates the zebrafish inflammasome and that this response is host protective, we next began to assess the host leukocyte response to inflammasome activation. To determine the inflammatory response to inflammasome activation, we infected embryos with fluorescently labelled macrophages or neutrophils and quantified the number of cells recruited to the initial site of infection. WT infection did not induce macrophage inflammation compared to mock inoculation with PBS. In contrast, Lmpyro infection elicited macrophage recruitment, resulting in an approximately 2-fold increase in the number of macrophages at the site of infection (Figure 5A–B). Neutrophil recruitment, on the other hand, was increased during both WT and Lm-pyro infections, independent of inflammasome activation (Figure 5C–D). Taken together, these data suggest that the acute inflammatory response to L. monocytogenes infection involves the recruitment of neutrophils whereas the macrophage compartment is specifically recruited during infection-induced inflammasome activation. Macrophages confer host protection to infection during inflammasome responses Given the specificity of the leukocyte response to bacterial infection with inflammasome activation, we next characterized the role of macrophages and neutrophils in controlling infection by depleting or severely impairing macrophage or neutrophil function, respectively. Knockdown of cell populations was carried out in transgenic lines labeling the relevant cell type(s) and verified by fluorescence microscopy immediately prior to experimentation. Cell Microbiol. Author manuscript; available in PMC 2017 April 01.

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Morpholino knockdown of the transcription factor Irf8 led to transient macrophage depletion until 2 days post infection in our model (Li et al., 2011). Control morphants were similar to WT larvae, as they were susceptible to WT infection and resistant to Lm-pyro (Figure 6A– B, 1A, 2A). Irf8 morphant fish, on the other hand, were highly susceptible to infection with both WT and Lm-pyro bacteria (Figure 6A–B). Indeed, macrophage deficiency eliminated the difference in survival outcome after infection in both control morphants and WT larvae (Figure 6G, 2A). This result was further verified using low dose Pu.1 morpholino injection as an alternative method to deplete macrophages but not neutrophils (Rhodes et al., 2005) (Supplementary Figure 4). In contrast, Δhly bacteria remained avirulent in both control and Irf8 morphants (Supplementary Figure 5A), suggesting that macrophage deficiency does not simply lead to general susceptibility to infection.


To address the role of neutrophils downstream of inflammasome activation we used a zebrafish model of the human disease leukocyte adhesion deficiency (Deng et al., 2011). Neutrophils expressing a dominant negative mutation in the small Rho family GTPase Rac2 have severely impaired neutrophil function (Deng et al., 2011). When expressed under a neutrophil specific promoter (Tg:mpx-Rac2 D57N), neutrophils enter the circulation but are unable to exit the vasculature and cannot reach localized sites of infection. Tg:mpx-Rac2 D57N embryos were more susceptible to both WT and Lm-pyro (Figure 6C–D). In contrast to macrophage depletion, however, Lm-pyro retained its virulence defect relative to WT bacteria when neutrophils were functionally defective (Figure 6G). As with macrophage deficiency, the virulence of Δhly was unaffected in Tg:mpx-Rac2 D57N larvae (Supplementary Figure 5B). Depletion of both macrophages and neutrophils by knockdown of the transcription factor Pu.1 (Rhodes et al., 2005) made larvae highly susceptible to infection, causing rapid death during WT, Lm-pyro, and even Δhly infection (Figure 6E–G and Supplementary Figure 5C), suggesting a total loss of host defense and extracellular replication of L. monocytogenes. Taken together, these findings suggest that macrophage and neutrophil responses are crucial in the response to L. monocytogenes infection. Specifically, the loss of macrophages re-sensitized larvae to infections with inflammasome activating bacteria, normalizing the virulence of WT vs Lm-pyro infections, whereas neutrophil deficiency generally increased susceptibility to L. monocytogenes infection but did not specifically rescue Lm-pyro virulence.

Discussion

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In the present study, we generated a new localized infection model to probe the role of the inflammasome in host defense to bacterial infection. We established that localized L. monocytogenes infection model requires the classic virulence factors Listeriolysin O and ActA for pathogenesis. Taking advantage of the genetic tractability of L. monocytogenes, we also showed that canonical inflammasome signaling during the response to Lm-pyro, a strain of L. monocytogenes engineered to ectopically secrete flagellin into the cytosol of host cells, leads to attenuation of virulence. Macrophages were specifically recruited in response to inflammasome activation, and blocking macrophage development rescued the virulence of Lm-pyro infections to WT levels. Neutrophils, on the other hand, were recruited to both WT and Lm-pyro infections. Although neutrophils were required for controlling both infections,


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functionally defective neutrophils did not rescue the virulence defect of Lm-pyro relative to infection with WT L. monocytogenes.

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L. monocytogenes has a wide natural host range in mammals, but has also been used as a model pathogen in alternative hosts including Danio rerio (zebrafish) (Menudier et al., 1996; Levraud et al., 2009; Shan et al., 2015), Drosophila melanogaster (fruit fly) (Mansfield et al., 2003), and Galleria mellonella (greater wax moth) (Mukherjee et al., 2013). In our localized infections, the classic virulence factors Listeriolysin O and ActA were required for virulence, in agreement with systemic infection of zebrafish larvae with L. monocytogenes (Levraud et al., 2009), and other invertebrate models (Mansfield et al., 2003; Mukherjee et al., 2013). At high enough doses ΔactA bacteria were virulent in our system, mirroring mouse models of listeriosis, where ΔactA mutants are lethal at an LD50 roughly 1000-fold above WT (Goossens and Milon, 1992). This further supports the idea that even within nonnatural hosts, L. monocytogenes’ ability to successfully complete the intracellular lifecycle within host cells is critical for virulence.

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Having demonstrated that our localized infection model requires the L. monocytogenes intracellular lifecycle for virulence, we sought to more fully establish the zebrafish larvae as a model system for examining inflammasome mediated responses. Our studies show that zebrafish are able to sense cytosolic flagellin and activate an inflammasome complex. In mice, flagellin is sensed by the Nlrc4 inflammasome in conjunction with the adaptor proteins NAIP5/6 (Kofoed and Vance, 2011; Zhao et al., 2011). Both Lm-pyro and S. typhimurium that overexpresses flagellin are severely attenuated in mice dependent on this sensor (Miao et al., 2010; Sauer et al., 2011). Humans have only one NAIP protein that does not recognize flagellin but rather senses type 3 secretion needle proteins (Yang et al., 2013). Mice have 6 NAIP proteins, some of which sense flagellin while other sense type 3 secretion inner rod proteins (Miao et al., 2010; Kofoed and Vance, 2011; Zhao et al., 2011; Rayamajhi et al., 2013). A large family of NLR family genes has been identified in the zebrafish genome, however, the presence of NAIP in zebrafish was unclear (Laing et al., 2008). Still, a number of related baculoviral inhibitor of apoptosis containing (BIRC) family proteins are present. Although our work does not identify specific NAIP or Nlrc4 homologue, it does provide strong evidence that, as in humans and mice, this signaling axis is present in the zebrafish immune system and is important for host defense against infection. Future studies that are able to match inflammasome ligands to their cognate sensors within the zebrafish immune system will strengthen the power of this system.

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Upon ligand sensing, the multi-protein inflammasome complex must form to achieve inflammatory outputs. Other studies in zebrafish larvae have also shown ASC and caspase A dependent effects during the response to inflammasome-associated stimuli including cholesterol induced gut inflammation (Progatzky et al., 2014), the response to viral infection (Varela et al., 2014), and tissue injury (Ogryzko et al., 2013). Using an ASC overexpression assay, we showed that ASC oligomerization increased only in response to Lm-pyro, but not to cytosolic bacteria alone. We also used a translation blocking morpholino to demonstrate that the attenuated virulence of Lm-pyro was dependent on caspase A, the zebrafish caspase-1 homologue (Masumoto et al., 2003). We were also able to witness downstream outcomes of inflammasome activation, including the entire process of macrophage


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pyroptosis in vivo at high temporal resolution. Hallmarks of pyroptosis have been seen in the zebrafish during the response to Shigella flexneri and SVCV infection (Mostowy et al., 2013; Varela et al., 2014). Although these studies were the first to show pyroptosis occuring in vivo, our approach expands on these findings. Through the use of fluorescently labelled histones, we were able to track nuclear morphology through the complete process of pyroptosis. Furthermore, increased temporal and spatial resolution allowed us to track cells through the process of pyroptosis, observing intermediate stages of death. Finally, to our knowledge, our work represents the first real time imaging of tissue resident macrophages undergoing pyroptosis. Thus, our data demonstrate full conservation of the canonical inflammasome in zebrafish: from ligand sensing by a conserved sensor, oligomerization of the core inflammasome component ASC, subsequent caspase-1 activation, and downstream outputs such as pyroptosis and control of bacterial infection.


Although it has been reported that wild type L. monocytogenes activate the NLRP3, NLRC4, and AIM2 inflammasomes in cell culture (Wu et al., 2010), the in vivo relevance of these findings has been controversial (Sauer et al., 2011). One early study suggested that Caspase-1 deficient mice are more susceptible to L. monocytogenes infection (Tsuji et al., 2004), however other studies have found only minimal inflammasome dependent phenotypes, both in alternative cell culture methods and in animal models (Sauer et al., 2011). Indeed, other pathogens such as S. typhimurium can activate the inflammasome experimentally but have evolved elegant mechanisms to avoid doing so during in vivo infection (Miao et al., 2010). We found no increase in macrophage recruitment or ASC speck formation over PBS inoculation following WT L. monocytogenes infection. Furthermore, there was no effect on the virulence of WT L. monocytogenes when caspase-1 was depleted, also consistent with some previous findings in mice (Sauer et al., 2011). Therefore our findings support the idea that L. monocytogenes, as well as other exquisitely evolved intracellular pathogens (Miao et al., 2010), largely avoids triggering the inflammasome to promote its virulence. However, a role for the inflammasome during WT L. monocytogenes infection cannot be entirely excluded.

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Few studies have examined the role of innate immune cells during the response to inflammasome activation in vivo. Macrophages have been used for much of the in vitro characterization of inflammasome activation, although dendritic cells have also been examined (Storek and Monack, 2015). Lm-pyro elicits an early recruitment of LysMpositive cells—monocytes or granulocytes—to the spleen whose numbers quickly drop (Williams et al., 2013). Studies in the mouse have also suggested a role for macrophages in carrying out inflammasome responses in vivo as S. typhimurium engineered to overexpress flagellin are found within macrophages undergoing pyroptosis (Miao, et al., 2010). In support of these studies, we found that macrophages were specifically recruited to inflammasome activation induced by Lm-pyro, and we observed infected macrophages undergoing pyroptosis in real time. Surprisingly, WT infection did not induce macrophage recruitment above mock inoculation. However, this is potentially due to the wound incurred during inoculation, as macrophage number in the HBV following PBS inoculation was higher than reported in equivalent stage embryos during normal development (Herbomel et al., 2001). Analysis of the kinetics of macrophage recruitment in this model will further clarify their role in the inflammatory response to L. monocytogenes infection, however our Cell Microbiol. Author manuscript; available in PMC 2017 April 01.

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findings suggest that inflammasome activation rapidly increases macrophage recruitment. Larvae lacking macrophages were equally susceptible to WT and Lm-pyro infection. We witnessed macrophage pyroptosis during Lm-pyro infection but were unable to witness pyroptosis during WT infection despite increased bacterial burden. Our findings, combined with previous reports (Miao et al., 2010; Sauer et al., 2011) suggest that macrophage pyroptosis is likely to be a critical inflammasome output for bacterial clearance after inflammasome activation. Whether macrophages function as sentinel cells that undergo pyroptosis to recruit effector cells or as direct antimicrobial mediators remains unknown.


Following macrophage death in mice, S. typhimurium overexpressing flagellin are taken up and killed by neutrophils in an NADPH oxidase-dependent manner (Miao et al., 2010), suggesting that neutrophils play an important role during the response to inflammasome activation. Neutrophil depletion did not, however, rescue the virulence defect of Lm-pyro in competitive index infections with WT L. monocytogenes (Sauer et al., 2011). In our experiments, neutrophils were recruited in equal number to WT and Lm-pyro infection. Furthermore, functionally defective neutrophils increased host susceptibility to both WT and Lm-pyro infection without removing the virulence defect of Lm-pyro. Therefore, while neutrophils were clearly important for controlling infection downstream of inflammasome activation, they were not required for inflammasome-dependent control of infection. Historically, the role of neutrophils in controlling L. monocytogenes has been unclear. Neutrophils have been deemed dispensable during L. monocytogenes infection because Ly6G antibody mediated depletion of neutrophils did not increase susceptibility to infection (Shi et al., 2011), but antibodies were given to mice after inoculation with bacteria. When applied before infection, Ly6G antibody mediated depletion does increase bacterial burden and host susceptibility as inoculating doses approached the LD50 (Carr et al., 2011). L. monocytogenes strains that are cytotoxic independent of the inflammasome are killed by neutrophils and rescued by neutrophil depletion, suggesting that L. monocytogenes is highly susceptible to killing by neutrophils (Glomski et al., 2003). In a recent study, Sox2 was identified as a neutrophil specific, cytosolic sensor of bacterial DNA that drives innate immune responses against L. monocytogenes (Xia et al., 2015). We found recruitment of neutrophils to the site of infection with L. monocytogenes, and a general increase in host susceptibility when neutrophil function was impaired. Our data therefore support an underappreciated role for neutrophils in the clearance of L. monocytogenes. Although this response was not specific to the inflammasome, understanding the role of neutrophils in response to L. monocytogenes infection is an ongoing focus of the lab.

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Since its initial characterization approximately 13 years ago (Martinon et al., 2002), the study of the inflammasome has quickly become a burgeoning field (Kanneganti, 2015). During this time, impressive advances have been made in understanding the molecular mechanisms of how the inflammasome senses pathogens and danger signals to drive inflammatory outputs. The interaction between pathogens and host cells during infection is complex, and the study of inflammasome signaling in vivo has been difficult. Model systems such as ours that display conserved inflammasome signaling and outputs, are genetically tractable, and are amenable to techniques such as high powered imaging will advance our understanding of how inflammasome activation and bacterial clearance are carried out by complex cell populations in vivo. Cell Microbiol. Author manuscript; available in PMC 2017 April 01.

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Experimental procedures Zebrafish Husbandry

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All zebrafish used in this study were on the AB background (originally received from the Zebrafish International Research Center). Previously published transgenic zebrafish lines Tg(mpeg1:dendra2) (Harvie et al., 2013), Tg(lyz:eGFP) (Hall et al., 2007), Tg(mpx:mCherry-2A-rac2wt) and Tg(mpx:mCherry-2A-rac2-d57n) (Deng et al., 2011) were used in this study. Adult zebrafish were maintained on a 14hr:10hr light/dark schedule. Upon fertilization, embryos were transferred into E3 buffer and maintained at 28.5°C. Before experimental manipulation, embryos were anesthetized using E3 buffer containing a final concentration of 0.2 mg/ml Tricaine (ethyl 3-aminobenzoate; Sigma Aldrich). All adult and larval zebrafish handling was carried out in full compliance of NIH guidelines and approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. Generation of transgenic Tg(mpeg1:mCherry-histone2b) zebrafish mCherry-histone 2B (von Dassow et al., 2009) flanked by XbaI and BamHI sites was cloned into the backbone vector with the mpeg1 promoter (Ellett et al., 2011) and minimal tol2 elements (Urasaki et al., 2006). Plasmid DNA and transposase mRNA were injected into early stage embryos, ~30–60 mpf. Generation of L. monocytogenes fluorescent reporter strains


To facilitate imaging ability, we constructed a set of fluorophore expressing strains of L. monocytogenes where fluorescence is driven either constitutively or downstream of the actA promoter. mCherry was codon optimized and synthesized as a gBlock gene fragment (Integrated DNA Technologies) flanked with EagI and SalI sites. The constitutive expression plasmid pPL2-GFP (Shen and Higgins, 2005) was digested with EagI and SalI, and EagImCherry-SalI was ligated in, generating a constitutive mCherry expression plasmid. From this plasmid, codon optimized mCherry was PCR amplified with ClaI and SalI flanking sites (forward primer TATTATatcgatATGGTTAGTAAAGGTGAAGAAGATAATATGGC.; reverse primer TTATAAgtcgacTTATTTATATAATTCATCCATACCACCTGTACTATGA) and ligated into ClaI and SalI digested actA promoter driven RFP expression plasmid (Waite et al., 2011). GFP expression driven by the actA promoter was constructed in an identical manner (forward primer TATTATatcgatATGAGTAAAGGAGAAGAACTTTTCACTGG; reverse primer TATTATgtcgacTTATTTGTATAGTTCATCCATGCCATGTGTAATC). In addition to these pPL2 constructs, constituve expression vectors were shuttled into pPL1 and pPL2e; actA driven expression vectors were shuttled into pPL2e. All primers were purchased from Integrated DNA Technologies. Bacterial culture and preparation All L. monocytogenes strains used were on the 10403s parental background. L. monocytogenes were grown in brain heart infusion (BHI) media. For infection experiments, cultures were grown statically in BHI overnight at 30°C to reach stationary phase. Bacteria were sub-cultured for ~1.5 hours in fresh BHI (4:1, BHI:culture) to achieve growth to mid-


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logarithmic phase (OD600 ≈ 0.5). Bacteria were washed three times in PBS, and resuspended to desired concentrations in PBS containing 10% glycerol and 2% PVP-40 (polyvinylpyrrolidine, Sigma Aldrich), to avoid bacterial clumping, and 0.3% phenol red for visualizing successful injections. Injection of bacteria into zebrafish larvae

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Manual dechorionation was performed using forceps at ~36 hpf. E3 media containing 3% agarose was polymerized in a petri dish on a shallow angle to make an injection ramp. At 48 hpf, anesthetized larvae were placed in the deep end of the injection ramp. For injections, batches of larvae were pulled to the shallow end of the ramp with a glass rod, such that E3 media with Tricaine just covered the larvae. One nl of bacterial solution was then directly injected into the HBV of embryos. Successful injections were monitored for the first minute post inoculation to verify that there was no leakage at the site of injection. Larvae were then transferred into 96-well flat bottom plates with fresh E3 lacking Tricaine to recover. For survival tracking, lack of a heartbeat was the readout for death. CFU enumeration from zebrafish larvae Larvae were injected as described above. At the indicated time points, larvae were homogenized by sheer stress in a bead beater, without beads added, in 200 μL of 1% saponin (Sigma) in PBS, in order to lyse host cells but leave bacteria intact. 50 μL of homogenate was plated and counted on LB agar with streptomycin (200 μg/mL; Sigma) using the Autoplate Spiral Plating System and QCount Colony Counter (Advanced Instruments). mRNA and morpholino injections


GFP-ASC mRNA was cloned into the pCS2+ vector. pCS2+ was digested with BamHI and XhoI (both Promega). BamHI-GFP-EcoRI was ligated to EcoRI-ASC-XhoI, which was then ligated into the backbone.mRNA was synthesized in vitro using the MAXIscript T7 kit (Ambion), and cleaned up using the RNeasy mini kit (Qiagen). Morpholino oligonucleotides (Gene Tools) were stored in 1 mM stock concentrations at room temperature. All mRNA and morpholino oligonucleotides were injected into early stage embryos, ~30–60 mpf, in a volume of 3 nl (pu.1, 500 μM for full knockdown of macrophages and neutrophils; 250 μM for macrophage only knockdown (Rhodes et al., 2005); irf8, 400 μM (Li et al., 2011); caspa, 250 μM (Masumoto et al., 2003); ASC-gfp mRNA, 100 ng/μl). For experiments using morpholinos to target the knockdown of phagocyte populations, experiments were carried out in the transgenic lines listed above, and phagocyte populations were verified immediately prior to the beginning of experiments by fluorescence microscopy. Phagocyte populations typically returned at 4 dpf. For caspase A knockdown, protein level was verified at 2 and 4dpf by Western blot (Supplementary Figure 3). Immunoblotting Whole protein lysates were taken from 50 larvae per sample for any given condition. 60 μg of lysate was loaded and run on an SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted for caspase A using a rabbit anti-zebrafish caspase A antibody (Anaspec). Actin was also immunoblotted as a loading control using a mouse anti-actin AC-15 antibody


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(Sigma). Blotting was imaged and quantified using an infrared Odyssey imaging system (LICOR). Fluorescence microscopy Anesthetized or 4% paraformaldehyde fixed embryos were imaged on custom made glass bottom imaging dishes. For live time lapse imaging, anesthetized larvae were stabilized by mounting in 1% low melt agarose with a final concentration of 0.2 mg/ml Tricaine as described above. Still image and time-lapse microscopy was performed via zoomscope microscopy (EMS3/SyCoP3; Zeiss; 1X PlanNeoFluar Z objective), laser scanning confocal microscopy (Fluoview FV1000; Olympus; 0.75 NA/20X objective), or spinning disk confocal microscopy (AxioObserver.Z1; Zeiss; 0.80 NA/20X or 1.30 NA/63X objective). Confocal images and movies used in figures were run through despeckling noise reduction in Fiji (Schindelin et al., 2012).

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Statistical analyses

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For survival curves, three independent experiments were performed. Results were pooled and analyzed by Cox proportional hazard regression analysis, with experimental conditions included as group variables. For CFU enumeration, three independent replicate experiments were conducted. CFU enumeration levels were compared between experimental conditions using analysis of variance. The results were summarized in terms of least squares adjusted means and standard errors. Graphical representation shows each individual data point. For ASC speck and leukocyte quantification, three independent experiments were performed. Results were analyzed by one way ordinary analysis of variance with a Tukey’s multiple comparisons test. For comparing differences of final survival outcomes, results were analyzed with Fisher’s exact test. For all tests a P value of < 0.05 was used to define statistical significance. Statistical analyses and graphical representations were all performed in R version 3 (R Development Core Team, 2013) or GraphPad Prism version 6.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments We thank members of the Sauer and Huttenlocher lab for fruitful discussions of the research and manuscript, and Jens Eickhoff for assistance with pooled statistical analyses. This work was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA188034 (JDS), National Institutes of Health Grant GM074827 (AH) and Molecular Biosciences Training Grant T32-GM07215 (WJBV).

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Author Manuscript Author Manuscript Author Manuscript Figure 1. L. monocytogenes virulence and dissemination in zebrafish larvae

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(A, B, C) Survival of larvae infected with WT, Δhly, and ΔactA, respectively at 10, 100, 1000, and 10000 CFU. (D, E, F) Dissemination of GFP expressing WT, Δhly, and ΔactA infection, respectively. (A, B, C) L. monocytogenes is virulent in zebrafish larvae, dependent upon the classic virulence factors listeriolysin O (hly) and ActA (actA). 48 hpf zebrafish larvae were infected with WT, Δhly, and ΔactA strains. Survival following inoculation was checked every 12 hours for 5 days. Each graph is the pooled data from three independent survival experiments. P-values and hazard ratios (HR) are relative to WT infection at the same dose of bacteria. (D, E, F) Representative images of dissemination over the course of 100 CFU infection. Larvae infected with WT, Δhly, and ΔactA strains constitutively


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expressing GFP were imaged daily. Δhly and ΔactA strains did not disseminate. Scale bars are 500 μm.

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Figure 2. Lm-pyro is attenuated in zebrafish larvae

(A) Survival of larvae infected with WT and Lm-pyro at 10, 100, 1000, and 10000 CFU. (B) Dissemination of GFP expressing WT and Lm-pyro. (A) Larvae were infected as in Figure 1. WT L. monocytogenes repeated with similar virulence. Lm-pyro displayed virulence defects at 10 and 100 CFU. At 1000 CFU virulence was equivalent, and slightly increased at 10000 CFU. Each graph is the pooled data from three independent survival experiments. Pvalues and hazard ratios (HR) are relative to WT infection at the same dose of bacteria. (B) Representative images of dissemination over the course of 100 CFU infection with WT and Lm-pyro. WT disseminated similar to Figure 1. Lm-pyro did not disseminate. Scale bars are 500 μm.

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Figure 3. Innate immune responses to Lm-pyro include ASC specks and pyroptosis

(A) Quantification of ASC specks at the site of infection and (B) representative images of larvae expressing GFP-ASC mRNA following 100 CFU infection. (C) Image stills (see also Supplementary Movie) showing macrophage pyroptosis during 100 CFU Lm-pyro infection. (A, B) Larvae were fixed at 12 hpi and imaged. ASC specks were quantified and normalized to the number of specks in PBS mock inoculated larvae. The graph is representative of three independent experiments. Scale bars are 100 μm. (C) Macrophages expressing the fluorophores dendra2 and mCherry in the cytosol and fused to histone-2B, respectively, were


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imaged. In macrophages undergoing pyroptosis, cytoplasmic signal was rapidly lost, coinciding with nuclear condensation. Scale bars are 10 μm.

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Author Manuscript Author Manuscript Figure 4. Lm-pyro virulence is rescued in the absence of caspase A

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Survival of morphant larvae infected with (A) WT and (B) Lm-pyro at 100 CFU. (A) WT virulence was unchanged in control or caspase A morphants. (B) Lm-pyro virulence was rescued during the depletion of caspase A. Each graph is the pooled data from three independent survival experiments.

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Figure 5. Phagocyte recruitment to WT and Lm-pyro infection

(A, B) Quantification of macrophages [Tg(mpeg1:dendra)] recruited to the HBV and representative images. (C, D) Quantification of neutrophils [Tg(lyz:eGFP)] recruited to the HBV and representative images. Larvae were inoculated with 100 CFU of bacteria, fixed at 6 hpi and imaged, and cells were quantified. Graphs are a single representative experiment from three independent experiments.

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Author Manuscript Author Manuscript Author Manuscript Figure 6. Macrophage depletion removes the virulence defect of Lm-pyro

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(A, B) Survival of Irf8 morphant larvae lacking macrophages following infection with WT and Lm-pyro, (A) and (B) respectively, at 100 CFU. (C, D) Survival of Tg(mpx-Rac2 D57N) larvae with functionally defective neutrophils following infection with WT and Lm-pyro, (C) and (D) respectively. (E, F) Survival of Pu.1 morphant larvae lacking both macrophage and neutrophil development following infection with WT and Lm-pyro, (E) and (F) respectively. Each graph is the pooled data from three independent survival experiments. Statistical analyses compare phagocyte impaired to non-impaired condition. (G) Comparison table displaying Fisher’s exact test comparisons, assessing the significant difference of final survival outcomes.


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Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin.

Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5.

Neutrophils in host defense: new insights from zebrafish.

Silica-induced inflammasome activation in macrophages: role of ATP and P2X7 receptor.

The role of lymph node sinus macrophages in host defense.

Posttranscriptional control of NLRP3 inflammasome activation in colonic macrophages.

mTORC1 Regulates Flagellin-Induced Inflammatory Response in Macrophages.

Strain- and host species-specific inflammasome activation, IL-1β release, and cell death in macrophages infected with uropathogenic Escherichia coli.

Activation of Cannabinoid Receptor 2 Ameliorates DSS-Induced Colitis through Inhibiting NLRP3 Inflammasome in Macrophages.

NLRP3 inflammasome activation mediates radiation-induced pyroptosis in bone marrow-derived macrophages.

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Chlamydia pneumoniae harness host NLRP3 inflammasome-mediated caspase-1 activation for optimal intracellular growth in murine macrophages.

NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes.

A role for host activation-induced cytidine deaminase in innate immune defense against KSHV.

NF-κB and P2X7R Signaling in PMA-Induced Macrophages.

Rhizobium meliloti nodD genes mediate host-specific activation of nodABC.

A20 overexpression alleviates pristine-induced lupus nephritis by inhibiting the NF-κB and NLRP3 inflammasome activation in macrophages of mice.

Temperature and host defense.

Inflammasome Activation Can Mediate Tissue-Specific Pathogenesis or Protection in Staphylococcus aureus Infection.

Gamma interferon, cytokine-induced macrophage activation, and antimicrobial host defense. In vitro, in animal models, and in humans.

LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis.

Monocytes.